Activated bifidobacteria and methods of use thereof

ABSTRACT

Some embodiments of the invention include a composition and method for treating dysbiosis in infants. The composition may include a mixture of activated bifidobacteria and a complex oligosaccharide wherein the complex oligosaccharide may be derived from a human or non-human source.

FIELD OF THE INVENTION

This invention relates generally to promoting health, and moreparticularly, to improving the human microbiome. Further, embodiments ofthis invention relate to activated bifidobacterial compositions, methodsof making activated bifidobacteria, and the use of the compositions inorder to initiate and maintain a human gut microbiome that is highlyenriched in Bifidobacterium species in order to facilitate thedevelopment of a human infant's gastrointestinal (GI) tract and immunefunction.

BACKGROUND

When a vaginally-delivered human infant is breast-fed he/she will have agastrointestinal microbiome that is unique in composition and diversitycompared to any other time in their life. The GI microbiome is dominatedby a single organism which can be present at high concentrations (up toand over 70% of the total microbiome). However, the obstetric standardof care in a typical hospital today involves births from both CesareanSection (C-section) and vaginal delivery, followed by human milk orinfant formula feeding for the baby. The surgical suite and levels ofcleanliness for the mother pre- and post-op in many modern hospitalsettings are such that in many cases the infant will not get seeded withbacteria normally found in the microbiome of the vagina orgastrointestinal tract of the mother, resulting in a dysbiosis in thebaby whether delivered by C-Section or vaginal births or fed by mother'smilk or infant formula. Furthermore, dysbiosis can also be caused byinfants losing the beneficial Bifidobacterium as a result of illness ormedical intervention (e.g., antibiotic treatment). The dysbiosis of theinfant microbiome leads to increased gastrointestinal problems anddelayed or altered immunological programming and tolerization. Theconsequences of early dysbiosis are considered to have an impactthroughout the entire life of that individual.

Human milk contains a significant quantity of complex oligosaccharides(up to 15% of total dry mass) in a form that is not usable as an energysource for the baby nor for most of the microorganisms in the gut ofthat baby. Certain microorganisms such as Bifidoacterium longum subsp.infantis [B. infantis or BI] have the unique capability to consume thespecific complex oligosaccharides such as those found in human or bovinemilk (U.S. Pat. No. 8,198,872 and U.S. Pub. No. 2013/0195803, thecontents of which are incorporated herein by reference). When B.infantis comes in contact with certain complex oligosaccharides a numberof genes are specifically induced within the bacterium whose proteinproducts as enzymes and binding proteins are responsible for the uptakeand internal deconstruction of those complex oligosaccharides, and theindividual sugar components are then catabolized to provide energy forthe growth and reproduction of that organism (Sela et al, 2008, PNAS,105(48): p. 18964-69).

SUMMARY

The instant invention provides compositions comprising isolated complexoligosaccharide fractions from mammalian milk sources, optionallysupplemented with purified fucosylated/sialylated oligosaccharides. Themammalian milk may be from human or bovine sources, and including butnot limited to, the bovine source is from bovine colostrum. Thefucosylated oligosaccharide(s) may comprise synthetically produced andpurified 2′-fucosyllactose, 3-fucosyllactose, difucosyllactose, orlacto-N-fucosylpentose.

In some embodiments, the composition further comprises bifidobacteriathat internalize the complex oligosaccharides or dietary glycans priorto their hydrolysis and metabolism. The combination of thebifidobacteria with the complex oligosaccharide may result in theconversion of the bifidobacteria to an activated bifidobacteria (ABI).The bifidobacteria is preferably selected from B. longum, B. breve, B.bifidum or B. pseudocatenulatum, and more preferably, the B. longum isB. longum subsp. infantis.

In other embodiments, any of the compositions described herein provide amethod of improving the health of a mammal comprising administering to amammal a composition comprising a complex oligosaccharide from amammalian milk source, optionally supplemented with a fucosylated and/orsialylated oligosaccharide, and a bifidobacteria that internalizes thecomplex oligosaccharide prior to its hydrolysis, and wherein thefucosylated oligosaccharide can be a synthetically produced and purifiedform of 2′-fucosyllactose, 3-fucosyllactose, difucosyllactose, orlacto-N-fucosylpentose, wherein the bifidobacteria is preferablyselected from B. longum, B. breve, B. bifdum or B. pseudocatenulatum,and more preferably, the B. longum is B. longum subsp. infantis. Thebifidobacteria is typically provided in a daily dose of from 10 thousandto 100 billion cfu, preferably 1 billion to 50 billion, and mostpreferably 5 billion to 25 billion, and the oligosaccharides areprovided in a daily dose of from 1 to 20 g, preferably in a daily doseof from 1 to 10 g.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram depicting whole genome expression analysis whichshows differential gene expression of B. infantis cells grown in thepresence of bovine milk oligosaccharides (BMO) or lactose.

FIG. 2 is a diagram showing a selection of the genes of the milkoligosaccharide cluster differentially expressed in B. infantis duringgrowth in the presence of bovine milk oligosaccharides (BMO) or lactose.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS I. Introduction

Human milk glycans contain a significant quantity of human milkoligosaccharides (designated herein as “HMOs”) (about 15% of total mass)in a form that is not usable as an energy source for the baby or formost of the microorganisms in the gut of that baby. HMOs can be found asfree oligosaccharides (dietary glycans) or conjugated to protein orlipids. The major HMOs in milk include lacto-N-tetraose (LNT),lacto-N-neotetraose (LNnT) and lacto-N-hexaose, which are neutral HMOs,in addition to fucosylated oligosaccharides such as 2-fucosyllactose(2FL), 3-fucosyllactose (3FL), and lacto-N-fucopentaoses I, II and III.Acidic HMOs include sialyl-lacto-N-tetraose, 3′ and 6′ sialyllactose(6SL). HMOs are particularly highly enriched in fucosylatedoligosaccharides (Mills et al., U.S. Pat. No. 8,197,872). Among theenzymes that produce HMOs in the mammary gland is the enzyme encoded bythe fucosyltransferase 2 (FUT2) gene, which catalyzes the linking offucose residues by an α1,2-linkage to oligosaccharides found in humanmilk. Fucosylated oligosaccharides are known to inhibit the binding ofpathogenic bacteria in the gut. HMOs, and in particular the fucosylatedHMOs, share common structural motifs with glycans on the infant'sintestinal epithelia known to be receptors for pathogens. (German etal., WO 2012/009315)

HMOs are substrates for the selective growth of certain bifidobacteriain the mammalian gut. Certain bifidobacteria such as, but not limitedto, Bifidobacterium longum subsp. infantis possess a gene clusterdedicated to the internalization and deconstruction of HMOs. When suchbacteria interact with HMOs, this gene cluster, including genes fortransporting and catabolizing fucosylated oligosaccharides, isupregulated. The interaction of certain HMOs with B. longum subsp.infantis has been shown to activate the bacterium by inducing expressionof a number of genes including, but not limited to, those in the HMOgene cluster that encode proteins to capture and internalize the HMOsand encode enzymes to completely catabolize the HMOs, thereby providingthat microbe with energy and substrate to grow and multiply (Underwood,2015, Pediatric Research, 77(1-2):229-35). The products of theupregulated genes also allow B. longum subsp. infantis to colonize themucosal lining of the gut and thereby impede the binding of pathogenicmicrobes. (Underwood, et al., 2015, Pediatr. Res., 77:229-235). Theactivated bacterium has been shown to possess increased binding tointestinal epithethial cells. (Chichlowski et al., 2012, J. PediatricGastroenteral Nur, 55:321-327). The activated B. longum subsp. infantisis also able to produce short chain fatty acids which facilitate thedevelopment of the infant's mucosal lining and the immune system(Underwood et. al., 2015, Pediatr Res, 77: 229-235). Consequently, theproliferation of ABI in the gut of a newborn infant, triggered anduniquely enabled by the HMOs provided in mother's milk, is ofsignificant benefit to the acute health and long term survival of thatinfant. Consequently, ABI provides significant benefits to a newborninfant which include, but are not limited to, a higher binding affinityto the gut mucosa, higher colonization of the GI tract therebypreventing growth of other bacterial clades, a higher production ofshort chain fatty acids, higher consumption of complex oligosaccharides,and a greater stimulation of the immune response as measured by positivealterations of immune response markers, relative to the organism in apre-activated state (Lewis, et al., 2015, Microbiome, 3:13; Huda, etal., 2014, Pediatrics, 134:2 e362-e372).

In the activated form, the B. infantis becomes the sole consumer of thehuman milk oligosaccharides (HMO) and has been shown to increase itsrelative proportion in the gut microbiota of infant humans to levels atleast 10-fold higher than its levels at birth (prior to consumption ofHMO), or in those infants exclusively fed commercial infant formula notcontaining milk oligosaccharides, and reaching levels as high as 70% ofthe total microbial population of the distal colon of breast-fed babies.When B. infantis is present in the gut of a baby, and that baby is alsoprovided with its mother's milk as a sole source of nutrition, thepopulation of B. infantis can increase to levels as high as 90% of thetotal bacterial population of the gut as measured by the microbialquantification of the stool. The ABI will remain in the gut at highconcentrations and remain activated as long as a dietary source of theselective complex oligosaccharides (e.g., HMO to human babies) isprovided to the infant. Once the source of the complex oligosaccharidesis withdrawn from the diet (e.g., at weaning and the introduction ofsolid foods), the B. infantis is no longer activated, it can no longersuccessfully compete other gut microbiota for nutrients in the gut, andits population decreases to less than 1% of the total microbiome. B.infantis is not normally found in the gut of a weaned infant, child, oradult in levels of more than 1% of the total microbiome

Surprisingly, B. infantis has also been shown to grow onoligosaccharides isolated from bovine milk (German, et al., WO2012/009315: Ward, 2009, Open Gyceroscience, 2:9-15). The concentrationof oligosaccharides in bovine milk oligosaccharides that are selectivein supporting the growth of B. infantis (designated as “BMO”) is low inmature bovine milk compared to that of human milk. This difference inabsolute concentration of oligosaccharides in milk may be due to aninitially low level of BMO in mature bovine milk or due to the presenceof enzymes in the milk that break down BMO into more simple sugars.Furthermore, the structural composition of the BMO is different fromthat of HMO (Aldredge et al., 2013, Glycobiology, (6):664-76; Mehra etal., 2014, PLoS One, 9(5):e96040). For example, BMOs are higher insialic acid-rich components than HMOs and lower in fucose-richcomponents than HMOs (Zivkovic and Barile, 2011, Advance Nutrition, (3):284-289).

Recently, the inventors have found that B. infantis and B. breve, whichare human-associated bacteria, are activated by bovine colostrumoligosaccharides (BCO), such as those found in a bovine colostrumoligosaccharide concentrate (BCOC). Colostrum is a special fluid thatcomes from the breast of mammals (e.g., humans and bovines) during thefirst few days after giving birth. The composition of colostrum issignificantly different from that of the mature milk which replaces thecolostrum after the first few days of lactation. Bovine colostrumoligosaccharides (BCOs) or bovine colostrum oligosaccharide concentrates(BCOs) have a composition that is different from HMO and BMO (U.S.Provisional Application No. 62/155,553, incorporated by referenceherein; Tao et al., 2009, Journal of Dairy Science, 92:2991-3001). Forexample, BCO is highly enriched in sialic acid residues but is deficientin many fucosylated oligosaccharides compared to mature milk BMO. Theinventors also discovered that there are a large number of genes inaddition to the HMO gene cluster that are either up-regulated ordown-regulated upon the interaction of B. longum subsp. infantis withthe different oligosaccharides. Some of these genes are regulated by HMOand some by BMO, BCO, or BCOC, and some, like the HMO cluster, areregulated by both.

II. Definitions

The term “oligosaccharide” refers to polymeric carbohydrates thatcontain 3 to 20 monosaccharides covalently linked through glycosidicbonds. In some embodiments, the oligosaccharides are purified from humanor bovine milk/whey/cheese/dairy products, (e.g., purified away fromoligosaccharide-degrading enzymes in bovine milk/whey/cheese/dairyproducts).

The term “isolated,” when applied to an oligosaccharide, refers to anoligosaccharide composition that has been at least enriched for theoligosaccharide compared to one or more other components in themammalian milk. In some embodiments, the oligosaccharide(s) is purified,e.g., such that the oligosaccharide has been separated at least in partfrom one or more of the other components of milk.

The term “bifidobacteria” and its synonyms refer to a genus of anaerobicbacteria having beneficial properties for humans. Bifidobacteria is oneof the major taxonomic groups of bacteria that make up the gut flora,the bifidobacteria are among the beneficial commensal bacteria thatreside in the gastrointestinal tract and have health benefits for theirhosts.

The term “synthetic” composition refers to a composition produce by achemi-synthetic process and can be nature-identical. For example, thecomposition can include ingredients that are chemically synthesized andpurified or isolated. This does not include compositions that arenaturally synthesized by mammals.

The term “residues,” when applied to an oligosaccharide, refers tomonosaccharide residues of oligosaccharides joined through glycosidiclinkages, which can be hydrolyzed by enzymes or acid to give theconstituent monosaccharide units.

III. Compositions

The compositions described herein comprise a non-pathogenic microbeand/or at least one complex oligosaccharide that induces a change in thenon-pathogenic microbe such that the complex oligosaccharide thenbecomes an energy source for the microbe, and when ingested by a mammal,the induced or activated microbe provides a benefit to the gut of thatmammal.

A. Complex Oligosaccharide

In various embodiments, the composition comprises a plurality ofoligosaccharides. The oligosaccharide composition may be derived fromhuman and non-human glycan sources and may exist as free glycans orprotein-bound glycans. In some embodiments, the oligosaccharide can be abovine or human milk oligosaccharide. In some embodiments, theoligosaccharide composition comprises bovine milk oligosaccharides(BMOs). Bovine oligosaccharides may comprise oligosaccharides frommature milk, early milk, colostrum, or concentrates thereof. In someembodiments, the oligosaccharides can include, but are not limited to,fucose, sialic acid, N-acetylglucosamine, and/or gluconate residues.

In various embodiments, complex milk oligosaccharides include anoligosaccharide consisting of 3 Hex moieties, 4 HexNAc moieties and 1fucose (Fuc) moiety; an oligosaccharide consisting of 4 Hex moieties, 4HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting of 3Hex moieties, 5 HexNAc moieties, and 1 Fuc moiety; an oligosaccharideconsisting of 5 Hex moieties, 4 HexNAc moieties, and 1 Fuc moiety; anoligosaccharide consisting of 4 Hex moieties, 5 HexNAc moieties, and 1Fuc moiety; an oligosaccharide consisting of 3 Hex moieties, 6 HexNAcmoieties, and 1 Fuc moiety; an oligosaccharide consisting of 3 Hexose(Hex) moieties and 6 N-acetyl hexosamine (HexNAc) moieties; anoligosaccharide consisting of 4 Hex moieties and 3 HexNAc moieties; anoligosaccharide consisting of 3 Hex moieties and 4 HexNAc moieties; anoligosaccharide consisting of 6 Hex moieties and 2 HexNAc moieties; anoligosaccharide consisting of 4 Hex moieties and 4 HexNAc moieties; anoligosaccharide consisting of 3 Hex moieties and 5 HexNAc moieties; anoligosaccharide consisting of 5 Hex moieties and 4 HexNAc moieties; anoligosaccharide consisting of 4 Hex moieties and 5 HexNAc moieties; andan oligosaccharide consisting of 3 Hex moieties and 6 HexNAc moieties.Exemplary oligosaccharides include Lacto-N-Tetraose,Lacto-N-Neotetraose, Lacto-N-Fucopentaose I, Lacto-N-Fucopentaose II,Lacto-N-Fucopentaose III, Lacto-N-Fucopentaose V, Lacto-N-Hexaose,Para-Lacto-N-Hexaose, Lacto-N-Neohexaose, Para-Lacto-N-Neohexaose,Monofucosyllacto-N-Hexaose II, Isomeric Fucosylated Lacto-N-Hexaose (1),Monofucosyllacto-N-Hexaose, Isomeric Fucosylated Lacto-N-Hexaose (3),Isomeric Fucosylated Lacto-N-Hexaose (2),Difucosyl-Para-Lacto-N-Neohexaose, Difucosyl-Para-Lacto-N-Hexaose,Difucosyllacto-N-Hexaose, Lacto-N-Neoocataose, Para-Lacto-N-Octanose,Iso-Lacto-N-Octaose, Lacto-N-Octaose, Monofucosyllacto-Nneoocataose,Monofucosyllacto-N-Ocataose, Difucosyllacto-N-Octaose I,Difucosyllacto-N-Octaose II, Difucosyllacto-N-Neoocataose II,Difucosyllacto-N-Neoocataose I, Lacto-N-Decaose,Trifucosyllacto-N-Neooctaose, Trifucosyllacto-N-Octaose andTrifucosyl-Iso-Lacto-N-Octaose.

In some embodiments, the oligosaccharide described herein comprisesthree or more monosaccharides (i.e., at least a trisaccharide), and canbe a bovine or human milk glycan, or the equivalent thereof that ischemically synthesized. The complex oligosaccharide may be, but is notlimited to, (3Hex,4HexNAc,1Fuc), (1Gal,1GcNAc,1NeuAc), and/or(1Glu,1Gal,1NeuAc (3′ or 6′)).

In some embodiments, the oligosaccharide described herein comprises anyof Hex(4); Hex(4) HexNAc(2); and Hex(3) HexNAc(1) NeuAc(1) at levelsgreater than 1%. In another embodiment, the at least one oligosaccharidecomprise one of the following ratios of constituents: 1) a ratio ofHex(2) NeuAc(1):Hex(2) HexNAc(1) less than 5.0; 2) a ratio of Hex(2)HexNAc(1):Hex (3) HexNAc(1) of greater than 1.0; 3) a ratio of Hex(2)HexNAc(1):Hex (3) HexNAc(2) of greater than 2.0; 4) a ratio ofHex(3):Hex (3) HexNAc(1) NeuAc(1) of less than 100; and 5) a ratio ofHex(2) HexNAc(1):Hex (4) NeuAc(2) NeuGc(1) of greater than 10.

Complex mammalian milk oligosaccharides (MMO) can be isolated from anynumber of sources and using methods known to those of skill in the art.For example, HMOs can be obtained from human milk using methods known inthe art. Human milk may be provided by the International Milk Bank(Sparks, Nev., USA) or any such equivalent milk bank. Human milk may bepasteurized and then centrifugally defatted, separating it into cream(predominantly fat) and skim (defatted product). The defatted skim milkmay then be filtered using membranes with a 5-10 kDa cut off toconcentrate a protein fraction (predominantly whey) and the permeate,comprising the complex HMOs. The composition of this dried HMO fractionis about 50% lactose and about 30% HMO with the remainder of the massprimarily peptides and ash. The HMO fraction is predominantlyfucosylated. The permeate may be further passed through a 1 kDa cut offfilter to remove lactose and provide a more enriched HMO fraction in theretentate prior to spray drying. BMOs can be isolated similarly, usingany number of sources and methods known to those of skill in the art.For example, BMO can be isolated using the purification protocols asdisclosed in the US Pub. No. 20130035481, the contents of which areincorporated herein by reference.

Colostrum oligosaccharides (COs) can be isolated from mammalian sourcessuch as, but not limited to cows (BCO), humans (HCO), goats (CCO), orsheep (OCO) and used in the instant invention. Colostrum can be used aswhole colostrum or processed to selectively enrich the CO fraction.Processing steps could include, but are not limited to, pasteurization,centrifugation, precipitation, ultrafiltration and spray drying. Ingeneral, the processes are selected to remove, inhibit or destroyenzymes that degrade the COs. In some embodiments, additional processingsteps can be used to sterilize the product to eliminate any potentialbacterial or viral contamination. Such steps include, but are notlimited to, conventional pasteurization, ultrahigh temperature (UHT)processes, gamma irradiation, freezing and thawing, sonication, andmicrofluidic disruption. In other embodiments, the lactose content ofthe BCO may be reduced using processes know in the art such as, but notlimited to, the treatment of the extract with enzymes to degrade lactoseor through mechanical or biological means of selective removal oflactose. In yet other embodiments of the invention, the liquid COmixtures are concentrated and/or dried by processes such as, but notlimited to, spray drying, freeze drying, fluid bed drying, tunneldrying, and drum drying.

In various embodiments, the complex oligosaccharide comprises at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or at least 95% of the dry weight of thecomposition.

In alternate embodiments, the complex oligosaccharide further comprisessynthetically produced oligosaccharides comprising fucosyllactose (SPF)and/or synthetically-produced sialyllactose (SPS) or derivatives thereofincluding, but not limited to, 2′-fucosyllactose, 3-fucosyllactose,difucosyllactose, lacto-N-fucosylpentaose I, lacto-N-fucosylpentaose II,lacto-N-fucosylpentaose III, lacto-N-fucosylpentaose V,3′-sialyllactose, 6′-sialyllactose, 3′-sialyl-3-fucosyllactose,sialyllacto-N-tetraose, and 6′-sialyllactosamine. The syntheticallyproduced oligosaccharides (SPO) may be derived using any of the numberof sources and methods known to those of skill in the art. For example,SPF is produced using protocols as disclosed in the US Pub. No.20130035481, the contents of which are incorporated herein by reference.

The synthetically-produced oligosaccharides (SPOs) can be added to thebiologically produced mammalian milk oligosaccharide (MMO) and make upfrom at least 5% to at least 80% of the dry weight of the composition.In some embodiments, the composition comprises a mixture of MMO and SPFand/or SPS. In various embodiments, the SPO is 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%of the dry weight of the composition. In some embodiments, the SPF is1-50% of the dry weight of the composition. In other embodiments, theSPO is 5-30% of the dry weight of the composition. In other embodiments,the SPO is 10-20% of the dry weight of the composition. The MMOcomprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the dryweight of the composition. In some embodiments, the MMO comprises BCOswherein the BCOs comprise at least 20% of the dry weight of thecomposition. In another preferred embodiment, the BCOs comprise at least50% of the dry weight of the composition. In another preferredembodiment, the BCOs comprise at least 70% of the dry weight of thecomposition. In some embodiments, the mass ratio of MMO:SPO is from 20:1to 1:10. In some embodiment, the ratio is from 10:1 to 1:2, and inanother embodiment, the ratio is from 5:1 to 1:1. In some examples, theratio is about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2 about 1:4,about 1:5, about 1:6 about 1:3, about 1:3, 10:2, about 9:2, about 8:2,about 7:2, about 6:2, about 5:2, about 4:2 or about 3:2.

B. Non-Pathogenic Microbes

In various embodiments, the composition comprises one or more species ofnon-pathogenic microbes, where one of the non-pathogenic microbes isfrom a species whose genome encodes a transport system capable ofinternalizing one or more complex oligosaccharides before theoligosaccharide is internally hydrolyzed. In various embodiments, themicrobe is from the genus Bifidobacterium. The species may be, but isnot limited to, B. longum, B. bifdum, B. breve, B. pseudocatenulatum, B.catenulatum, or any Bifidobacterium strain that expresses a fucosidase,or any combination of these Bifidobacterium species. In someembodiments, one of the species is Bifidobacterium longum and inpreferred embodiments, one or both of the species is Bifidobacteriumlongum subspecies infantis.

In various embodiments, the Bifidobacterium may comprise activatedBifidobacterium (ABI). The activated Bifidobacterium is defined hereinas the state of the cells, as measured by the up-regulation ordown-regulation of genes including but not limited to those coding foroligosaccharide binding proteins, transport proteins, and enzymesresponsible for the degradation of the complex oligosaccharides, whichprovides significant benefits to a newborn infant. Such beneficialcharacteristics of the ABI include, but are not limited to, a higherbinding affinity to the gut mucosa, higher colonization of the GI tractthereby preventing growth of other bacterial clades, a higher productionof short chain fatty acids, the ability to consume complexoligosaccharides, and a greater stimulation of the immune response asmeasured by positive alterations of immune response markers, relative tothe organism in a pre-activated state (Lewis, et al., 2015, Microbiome,3:13; Huda, et al., 2014, Pediatrics, 134:2 e362-e372).

In various embodiments, the bifidobacteria encodes gene clusterscontaining ATP-binding cassette (ABC) transporters and glycosylhydrolases involved in HMO utilization, typically including one or moregenes coding for a fucosidase. In some embodiments, the bifidobacteriacontains a gene coding for a complex oligosaccharide transporter. Insome embodiments, the bifidobacteria contains a gene coding for a fucosetransporter. In some embodiments, the bifidobacteria contains a genecoding for a fucose or sialic acid transporter. In many embodiments, thegenes encoding these components are upregulated or expressed. The genesmay be constitutively upregulated or induced.

Certain biomarkers may be induced and/or repressed as markers to predictan activated state for bifidobacterium species, whereby the bacteria areoptimally primed for complex oligosaccharide consumption. Suitablebiomarkers identified with B. longum subsp. infantis activation includeupregulated genes and downregulated genes. Exemplary upregulated genesinclude Blon_0042 (regulatory protein); Blon_R0015 (tRNA), Blon_R0017(tRNA); Blon_R0021 (tRNA); and Blon_R0022 (tRNA). Exemplarydownregulated genes include Blon_0518 (hypothetical protein); Blon_0785(membrane lipoprotein (possible transporter component)); Blon_2167(hypothetical protein); and Blon_2168 (phage shock protein C).Previously, these genes were not known to be associated with anactivated cell.

In some embodiments, the activated bifidobacteria comprises geneBlon_0042, wherein gene Blon_0042 has been upregulated. The activatedbifidobacteria may comprise gene Blon_2168, wherein gene Blon_2168 hasbeen downregulated. In one embodiment, the activated bifidobacteriacomprises gene Blon_0042 and gene Blon_2168, wherein gene Blon_0042 hasbeen upregulated and gene Blon_2168 has been downregulated. The skilledperson can readily adapt quantitative proteomic methods to determine theexpressed levels of the gene products (e.g., mRNA and protein) for thesegenes, to confirm activation.

ABI is activated by being cultivated in a medium comprising at least oneoligosaccharide among the complex oligosaccharides described above for asufficient period of time to undergo induction and biosynthesis of atleast one metabolic enzyme. The oligosaccharides are typically sourcedfrom, or are identical to, those mammalian milk oligosaccharides (MMOs)including, but not limited to, those from human milk and bovine milk. Insome embodiments, the oligosaccharide can be a bovine or human milkoligosaccharide. In another embodiment, the oligosaccharide is obtainedfrom mammalian colostrum. In some embodiments, the oligosaccharidecomposition comprises bovine milk oligosaccharides (BMOs). Bovineoligosaccharides may comprise oligosaccharides from mature milk, earlymilk, colostrum, or concentrates thereof. In some embodiments, theoligosaccharides include fucose as component saccharide residues. In analternative embodiment, the MMO is supplemented with syntheticallyproduced and purified oligosaccharides comprising fucosylated and/orsialylated oligosaccharides. In some embodiments of the invention, thesynthetically-produced fucosyllactose (SPF), sialyllactose (SPS) orderivatives thereof are used to activate bifidobacteria in a way that ismore human-like than when activated by BMOs alone. In anotherembodiment, the composition is used to upregulate operons other than theHMO cluster.

Any of the compositions described herein may be prepared by cultivatinga bifidobacteria in an axenic culture (e.g., a culture with genetichomogeneity), the culture comprising bovine milk glycans, (e.g.,concentrated from bovine colostrum) to become “activated.” In variousembodiments, any of the compositions described herein can be made byisolating bifidobacteria; purifying the bacteria; inoculating afermenter with the purified strains of the bifidobacteria; and culturingthe bifidobacteria in the presence of complex bovine or humanoligosaccharides; and harvesting the cells. Fermentations forbifidobacteria may be carried out in stirred tank fermenters ofcommercial volume (e.g., 1-500 m³) which are maintained under anaerobicconditions throughout the fermentation process. The fermentation caninclude the steps of providing at least one complex oligosaccharide atany time during the course of the fermentation in a liquid culture at alevel of at least 1 g/L, typically from about 1-50 g/L, or 2-20 g/L, or5-10 g/L as a sole, or supplementary, carbon source to activate thecells.

The bifidobacteria described herein may be tested for its ability to usebovine or human milk oligosaccharides for growth. In some embodiments,the bifidobacteria are capable of growing on mammalian milk glycanswhere less than 20% of the sialic acid content and 20% of the fucosecontent of the milk glycans remains after a culture of the compositionhas ceased to grow. In some embodiments, the composition is capable ofgrowing on mammalian milk oligosaccharide wherein less than 10% of thesialic acid content and 10% of the fucose content of the milk glycansremains after a culture of the composition has ceased to grow. In apreferable embodiment the composition is capable of growing on milkglycans wherein less than 5% of the sialic acid and 5% of the fucose ofthe milk oligosaccharides remains after a culture of the composition hasceased to grow. In a particularly preferable embodiment, the compositionis capable of growing on milk glycans wherein less than 1% of the sialicacid and 1% of the fucose of the milk oligosaccharides remains after aculture of the composition has ceased to grow.

In a further embodiment, the composition can comprise a total count ofviable bacteria from about 100 thousand to 500 billion colony formingunits (cfu) per gram dry weight. In another embodiment, the total countof viable bacteria comprises 5 billion to 100 billion cfu per gram dryweight. In another embodiment, the total count of viable bacteriacomprises 10 billion to 50 billion cfu per gram dry. In someembodiments, the ABI concentration is from 10 to 100 g dry weight perliter. The fermentation products can also be concentrated by filtrationor centrifugation. The ABI can be can be dried by controlled desiccationprocesses such as, but not limited to, freeze drying.

IV. Formulating Compositions

The composition comprising MMO and ABI can be prepared by mixing the twocomponents together. Optionally, one can combine the harvested and/ordried activated bifidobacteria cells with a powdered form of a complexbovine or human milk oligosaccharide. The harvested and/or driedactivated bifidobacteria cells and the powdered form of the complexbovine or human milk oligosaccharide can be in a single dose packet,which can contain from about 10 million to about 100 billion cfu ofbacteria and, optionally, from about 0.5 g to about 5 g of complexoligosaccharide. The complex bovine oligosaccharide can be present in apowder composition wherein the blend ratio of activated bifidobacteriacells to complex oligosaccharide is 30 billion cfu per 1.5 g complexoligosaccharide in a powder form.

Any of the compositions described herein can further comprise asecondary metabolite. The secondary metabolite can be a short chainfatty acid, such as acetate, lactate, or combinations thereof. Thecompositions described herein can further comprise a stabilizer, such asa flow agent. Flow agents may include starch, silicon dioxide,tricalcium phosphate, powdered cellulose, magnesium stearate, sodiumbicarbonate, sodium ferrocyanide, potassium ferrocyanide, calciumferrocyanide, bone phosphate, sodium silicate, calcium silicate,magnesium trisilicate, talcum powder, sodium aluminosilicate, potassiumaluminum silicate, calcium aluminosilicate, bentonite, aluminumsilicate, stearic acid, and polydimethylsiloxane. The stabilizer can bea milk protein or another suitable pharmaceutical grade or infantformula grade diluent (e.g., lactose). The milk protein can comprise aprotein fraction of non-fat dry milk.

Any of the compositions described herein can further comprise surfacecarbohydrate binding protein (e.g., solute binding proteins). Thesurface carbohydrate binding proteins can allow a more effective bindingand interaction with the gut mucosa by binding to cell surfaceglycosylation of the gut mucosa and or mucous layers. This binding ofsurface carbohydrate can then exclude the binding of pathogenicbacteria.

In various embodiments, any of the compositions described herein may bedried (e.g., by spray-drying or freeze-drying), and formulated into aunit dose medicament, such as a packet, sachet, orally disintegratingtablet, foodstuff, capsule, lozenge, effervescent tablet, etc. The unitdose medicament can be formed from a variety of materials includingwithout limitation plastic, or paper. In some embodiments, the unit dosemedicament comprises a moisture barrier and/or oxygen barrier layer.Alternatively, the composition may be provided in a form for analdelivery, such as a suppository or in an enema. Preferably, thecomposition is packaged in sachets made using a moisture and/or oxygenimpermeable polymer.

In various embodiments, any of the compositions described herein may beprovided in a dry powder formulation, a solution, a suspension, or in atablet or capsule format with or without an enteric coating. The drypowder can be freeze-dried or spray dried. The freeze-dried compositionsare preferably frozen in the presence of a suitable cryoprotectant. Thecryoprotectant can be, for example, glucose, lactose, raffinose,sucrose, trehalose, adonitol, glycerol, mannitol, methanol, polyethyleneglycol, propylene glycol, ribitol, alginate, bovine serum albumin,carnitine, citrate, cysteine, dextran, dimethyl sulphoxide, sodiumglutamate, glycine betaine, glycogen, hypotaurine, peptone, polyvinylpyrrolidone, or taurine. The enteric coatings include, but are notlimited to, fatty acids, waxes, shellac, plastics, plant fibers, methylacrylate-methacrylic acid copolymers, cellulose acetate succinate,hydroxy propyl methyl cellulose phthalate, hydroxy propyl methylcellulose acetate succinate, polyvinyl acetate phthalate (PVAP), methylmethacrylate-methacrylic acid copolymers, cellulose acetatetrimellitate, sodium alginate, and Zein.

In some embodiments, the microbe is mixed with a cryopreservative suchas but not limited to trehalose or glycerol under anaerobic conditionsand frozen by processes such as, but not limited to, rapid freezing(chilling with liquid nitrogen), or by a controlled temperaturereduction in a cryopreservation freezing system. Once frozen, themicrobes can be dehydrated under vacuum using a process that bestmaintains the integrity of the microbe cells. The microbe concentrationin the dry powder can be from 1 million to 500 billion cfu/g. In someembodiments, the dry powder can be from 5 billion to 100 billion cfu/g,and in a most preferred embodiment the dry powder can be from 10 billionto 50 billion cfu/g.

In some embodiments of the invention, the powdered microbe isresuspended in an edible oil such as, but not limited to triglycerideoils (e.g., vegetable oil, olive oil, and medium chain triglycerides),diglyceride oils, monoglyceride oil, and silicone oils.

In various embodiments, the oligosaccharide composition can be dissolvedin a polar liquid such as, but not limited to, water, physiologicalsaline, mammalian milk, or an infant formula, and provided in a liquidform to the infant while the bifidobacteria are provided separately as apowder or suspension in a carrier liquid which may include a solutioncomprising the oligosaccharide.

In various embodiments, the microbes and the oligosaccharide compositionmay be provided combined or provided separately. In some embodiments,the microbe is combined with an oligosaccharide in a single dose packetcontaining from about 1 to about 100 billion cfu of microbe and fromabout 0.5 to about 5 g of an oligosaccharide.

V. Use of Compositions for Improvement of Mammalian Health

In various embodiments, the compositions described herein are deliveredas a pre-activated and purified composition of bifidobacterium to asubject in need thereof substantially contemporaneously with delivery ofcompounds to the mammalian intestine to make the intestinal environmenta more favored niche to the aforementioned purified composition ofbifidobacteria, where the compounds may comprise complexoligosaccharides described above, synthetically produced and purifiedoligosaccharides, and/or secondary metabolites produced as a result ofintestinal fermentation.

In various embodiments, the use described herein comprises monitoringthe subject's intestinal microbiome before, during and/or afteradministration of the composition described herein. A variety ofmonitoring techniques are known to one of ordinary skill in the art. Forexample, a routine sample of the subject's feces may be analyzed formicrobes qualitatively and/or quantitatively by standard processes wellknown in the art (see, e.g., Le Pare et al., 2014, Food and NutritionSciences, 5: p. 71-78).

In some embodiments, the compositions described herein are administeredto a subject in need thereof in an amount and for a duration effectiveto establish the population of bifidobacteria at high levels in thegastrointestinal tract of the subject. In some embodiments, thecompositions described herein can be administered to a subject in needthereof in an amount and for a duration effective to maintain thepopulation of bifidobacteria at high levels in the gastrointestinaltract of the subject. In some embodiments, the composition isadministered daily in an effective amount to maintain the bifidobacteriapopulation in the gut of the subject at greater than at least 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% of the total fecalmicrobiome of the mammal.

In some embodiments, the composition comprising activated bifidobacteriais administered to a subject in need thereof. In other embodiments, thecomposition comprising complex oligosaccharides supplemented withsynthetically produced and purified oligosaccharide is administered to asubject in need thereof. In another embodiment, the compositioncomprising both activated bifidobacteria and complex oligosaccharide isadministered to a subject in need thereof. In another embodiment, thecomposition comprising bifidobacteria, and complex oligosaccharidessupplemented with synthetically produced and purified oligosaccharide isadministered to a subject in need thereof.

In various embodiments, the bifidobacteria are administered at a dose offrom 1 billion to 100 billion cfu of bifidobacteria and from 1 to 20 gof complex oligosaccharides per day. In some embodiments, a dose isadministered from 5 to 50 billion cfu/day. In another embodiment, a doseis administered from 5 to 100 billion cfu/day. In another embodiment,the dose is administered from 10 to 25 billion cfu/day. In variousembodiments, the complex oligosaccharide is administered in a dose offrom 0.5 g to 5.0 g/day. In some embodiments, the dose is administeredin a dose from 1.0 g to 3.0 g/day.

Typically, the composition of this invention is presented as a single,unit dose package that is administered once per day. However, the dosesmay be presented in multiple (e.g., two, three, four, five, six, ormore) sub-doses administered at appropriate intervals throughout theday. Alternatively, they may be administered in the same composition, orconstituent components may be administered sequentially. In someembodiments, the treatment is maintained for a period of at least1-week, 2-weeks, 3-weeks, or at least 4-weeks. In other embodiments, thetreatment is administered for a period of from at least 2-months,4-months, 6-months, 8-months, 10-months, or at least 12-months.

The subject in need thereof can be, for example, an infant from birth toabout 36 months post-conception. In additional embodiments, thecompositions described herein may be administered to a pregnant woman inat least the third trimester of pregnancy. The composition administeredduring pregnancy may include either the bifidobacteria, theoligosaccharide, or both. In additional embodiments, the compositiondescribed herein is administered in a therapeutic amount to an infantborn vaginally or by cesarean section. The compositions described hereinare administered to the infant immediately after delivery and thereafterfor at least the first month to six months of the life of the infant.The composition may be administered directly to the infant or mixed witha liquid including, but not limited to breast milk, infant formula,physiological saline, or water. For infants who are not breast fed, thecompositions described herein may alternatively be administered in aninfant formula and such compositions may preferably comprise bothactivated B. infantis and a milk-derived oligosaccharide. For infantsborn via cesarean section, compositions comprising of activatedbifidobacteria and/or complex oligosaccharides may be administered. Forinfants born vaginally, compositions comprising activated bifidobacteriaand/or complex oligosaccharides may be administered.

The above-described embodiments of the invention are presented forpurposes of illustration and not of limitation. While these embodimentsof the invention have been described with reference to numerous specificdetails, one of ordinary skill in the art will recognize that theinvention can be embodied in other specific forms without departing fromthe spirit of the invention. Thus, one of ordinary skill in the artwould understand that the invention is not to be limited by theforegoing illustrative details, but rather is to be defined by theappended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1. Preparation of Human Milk Oligosaccharide (HMO) Compositionsthat can be Used Exclusively by Certain Bifidobacteria

A concentrated mixture of HMO is obtained by a process similar to thatdescribed by Fournell et al (US Patent Application 20150140175). Humanmilk is pasteurized and then centrifugally defatted, separating it intocream (predominantly fat) and skim milk (defatted product). The defattedskim milk is then ultrafiltered using membranes with a 5-10 kDa cut offto concentrate a protein fraction (predominantly whey). The permeatefrom the ultrafiltration, comprising the complex HMOs, is dried by spraydrying. The composition of this dried fraction is about 50% lactose andabout 30% complex oligosaccharides (HMO) with the remainder of the massprimarily peptides and ash. The HMO fraction is predominantlyfucosylated.

This method or similar methods can be used to obtain compositionscontaining isolated complex oligosaccharides from any mammalian milksource. For example, complex oligosaccharides can be isolated using thepurification protocols as disclosed in the US Pub. No. 20130035481, thecontents of which are incorporated herein by reference. Suchcompositions are suitable for use in embodiments of this invention.

Example 2. Preparation of Bovine Colostrum Oligosaccharide (BCO)Compositions and Compositions Supplemented with Synthetically Producedand Purified Fucosylated Oligosaccharides (SPF) that can be UsedExclusively by Certain Bifidobacteria

A concentrated mixture of bovine colostrum oligosaccharide is obtainedby a process such as that described by Christiansen et al (2010)International Dairy Journal, 20:630-636. Bovine colostrum (preferablyfrom the first milking) is pasteurized by heating to 145 degrees F. for30 minutes, cooled and centrifugally defatted, separating it into cream(predominantly fat) and skim milk (defatted product). The defatted skimmilk is then ultra-filtered using membranes with a 5-10 kDa cut off toconcentrate a protein fraction (predominantly whey). The whey permeateis further microfiltered using a 1 kDa cut off to remove some lactoseand concentrate the oligosaccharides in the retentate. The finalcomposition is spray dried to produce a dried oligosaccharide fractionhaving about 40% lactose and about 40% complex oligosaccharides (BCO)with the remainder of the mass primarily peptides and ash. The BCOfraction is predominantly sialylated.

Synthetically produced and purified fucosylated oligosaccharides (SPF)can be can be obtained commercially from any of the number of sources orderived by methods known to those of skill in the art. 100 g of purified3-Fucosyllactose (SPF; Elicityl S A, Crolles, F R) may be added to 1 kgof the BCO preparation and thoroughly mixed to produce a BCO/SPFcomposition with a ratio of BCO:SPF of about 4:1. The sample is analyzedand the complex oligosaccharide component is about 20% fucosylatedoligosaccharides.

This method or similar methods can be used to obtain compositionscontaining complex oligosaccharides from any mammalian milk sourcesupplemented with particular synthetic oligosaccharides. Suchcompositions are suitable for use in embodiments of this invention.

Example 3. Differentiation Between BCO, BCOC, BMO and HMO Fractions

A BCOC fraction was commercially obtained as Immunel, (SterlingTechnology, USA) and analyzed using HPLC-MS methods of Tao et al (2008)J Dairy Science, 92: 2991-3001 and compared in Table 1 witholigosaccharide fractions from human milk (HMO), mature bovine milk(BMO) and bovine colostrum (BCO). The four compositions in Table 1 aresignificantly different from each other and several features areimmediately apparent. BCOC contains several oligosaccharides that arenot found in BMO, BCO or HMO such as Hex(4) and Hex(3) HexNAc(1)NeuAc(1), and several oligosaccharides that are found in BCO and BMO arenot present in BCOC such as Hex(2) HexNAc(1) NeuAc(1), Hex (3)HexNAc(2), and Hex (4) NeuAc(2) NeuGc(1).

TABLE 1 Key oligosaccharides that differentiate colostrum and BMOcompositions as disclosed in Tao (2009), HMO compositions as disclosedin Mills et al (2012) and bovine colostrum oligosaccharide concentrate(BCOC; Immunel) of the instant invention. Values listed are percentagesof the total sample oligosaccharides. m/z reduced BCO BMO neutral HexHexNAc NeuAc NeuGc Fuc Tao BCOC Tao HMO 506.1833 3 2.27 22.70 18.70 1.48547.2198 2 1 2.62 17.10 1.59 0.01 635.2263 2 1 20.33 26.00 22.61 1.82667.2300 4 0.10 1.40 0.01 0.01 676.2533 1 1 1 4.47 3.90 1.01 0.01709.2621 3 1 4.15 6.90 10.00 20.90 750.2892 2 2 5.62 3.30 0.01 0.01797.2772 3 1 4.98 3.20 23.11 0.01 838.3056 2 1 1 3.34 0.01 0.61 0.01855.3221 3 1 1 0.01 0.01 0.01 0.70 871.3153 4 1 7.35 7.50 12.46 0.01912.3409 3 2 4.73 0.01 2.85 0.01 942.3234 2 1 1 11.48 0.01 0.01 0.01999.3517 3 1 1 0.01 1.30 0.01 0.01 1074.3960 4 2 0.01 4.50 0.52 0.411177.4000 4 1 1 2.63 0.01 0.26 0.01 1220.4540 4 2 0.01 0.01 0.01 25.201366.5120 4 2 2 0.01 0.01 0.01 13.50 1439.5290 5 3 0.01 0.01 0.01 2.001585.5870 5 3 1 0.01 0.01 0.01 5.90 1731.6450 5 3 2 0.01 0.01 0.01 7.201804.6610 6 4 0.01 0.01 0.01 1.40 1877.7030 5 3 3 0.01 0.01 0.01 5.001950.7190 6 4 1 0.01 0.01 0.01 2.90 2096.7770 6 4 2 0.01 0.01 0.01 3.602242.8350 6 4 3 0.01 0.01 0.01 1.80

In addition, the ratios of the various bovine oligosaccharides to eachother are also quite different among the bovine sources as demonstratedin Table 2. The ratio of Hex(3) to Hex(3) NeuAc( ) of BCOC is greaterthan 1.0, the ratio of Hex(3) to Hex(4) HexNAc(2) of BCOC is less than20. and the ratio of Hex(2) HexNAc(1) to Hex(4) of BCOC is less than 20.Further, these bovine oligosaccharide compositions are all distinguishedfrom HMO in that in addition to the ratio differences all bovineoligosaccharide samples are further characterized by being less than 50%fucosylated. In addition, about 70% of bovine early milkoligosaccharides are sialylated in contrast to 50% in BMO, HMO, andN-glycolylneuraminic acid, which made up 7% of the sialic acid in thebovine early milk is completely absent in mature BMO (Tao, 2009). Evenwithout specifying fucosylation or sialylation, there are some ratios ofcomplex oligosaccharides that are unique in the BCOC of the presentinvention even when considering all bovine and human oligosaccharides.Such unique BCOC oligosaccharide signatures include; 1) a ratio ofHex(2) NeuAc(1):Hex(2) HexNAc(1) less than 5.0; 2) a ratio of Hex(2)HexNAc(1):Hex (3) HexNAc(1) of greater than 1.0; 3) a ratio of Hex(2)HexNAc(1): Hex (3) HexNAc(2) of greater than 2.0; 4) a ratio ofHex(3):Hex (3) HexNAc(1) NeuAc(1) of less than 100; and 5) a ratio ofHex(2) HexNAc(1):Hex (4) NeuAc(2) NeuGc(1) of greater than 10 (Table 2).

TABLE 2 Ratios of specific oligosaccharides found in BCO, BCOC, BMO andHMO from Table 1 Oligosaccharide ratios BCO BCOC BMO HMO Hex(2)NeuAc(1):Hex (2) HexNAc(1) 7.8 1.5 14.2 182.0 Hex(2) HexNAc(1):Hex (3)0.6 2.5 0.2 0.0 HexNAc(1) Hex(2) HexNAc(1):Hex (30 0.6 1710.0 0.6 1.0HexNAc(2) Hex(3):Hex (3) HexNAc(1) NeuAc(1) 227.0 17.5 1870.0 148.0Hex(2) HexNAc(1):Hex(4) NeuAc(2) 1.0 1710.0 6.1 1.0 neuGc(1)Hex(3):Hex(3) NeuAc(1) 0.5 7.1 0.8 148.0 Hex(3):Hex(4) HexNAc(2) 227.05.0 36.0 3.6 Hex(2) HexNAc(1):Hex(4) 26.2 12.2 159.0 1.0 Fucosylatedoligosaccharides (% 0.00 0.00 0.00 0.00 of total)

This example demonstrates that HMO, BMO, BCO, and BCOC have distinctlydifferent compositions. However, all four of these mixtures are able toactivate B. infantis.

Example 4. Preparation of an Activated Bifidobacteria (ABI) Compositionthat can Exclusively Use Certain Complex Oligosaccharides

Bifidobacteria longum subsp. infantis (alternatively B. infantis herein)was isolated and purified from the feces of a vaginally delivered,breast fed human infant and its identification was confirmed by DNAanalysis that reflected the presence of a gene set that is specificallyassociated with this organism (Sela et al., 2008, PNAS, 105(48): p.18964-69). Alternatively, a strain of B. infantis can be obtained from acommercial culture collection such as the American Type CultureCollection (ATCC) of Washington, D.C.

A seed culture of this organism was added to a growth medium comprisingglucose and a BCO composition, made using the process described inExample 2, and other standard salts and vitamins in a 500 L agitatedfermenter. Following 3 days of growth under anaerobic conditions, asample of the culture was tested for the presence of ABI. ABI wasidentified by the presence of expressed gene transcripts for fucosidaseor sialidase. The fermenter was harvested by centrifugation and theconcentrated cell mass was mixed with a cryopreservative (e.g.,trehalose plus milk proteins) and freeze dried. The final dry productwas 5.5 kg of bacterial mass with a count of 130×10⁹ cfu/g.

This example demonstrates that bifidobacteria can be activated byculturing the bifidobacteria with a complex bovine milk oligosaccharide.While BCO was used herein, this method or a similar method can be usedto obtain ABI by culturing with MMO from any mammalian milk. Such ABIwould be suitable for use in embodiments of this invention.

Example 5. Bifidobacteria Grown on Complex Oligosaccharides is Activatedfor Consumption of Milk Oligosaccharides

B. infantis ATCC 15697 was grown in MRS broth containing 2% lactose orbovine milk oligosaccharides (BMO). Cells were collected at exponentialphase, RNA was purified and converted to cDNA and sequenced on anIllumina platform. Results clearly show differential expression duringgrowth on BMO.

FIG. 1 depicts whole genome expression analysis. The diagram showsprinciple component analysis of all expressed genes within B. infantis.The diagram clearly shows differential expression of cells grown on BMOversus cells grown on lactose. 577 genes are differentially expressedsuggesting growth on milk oligosaccharides induces a differentphysiological state in B. infantis than lactose.

Further analysis shows that the 40 kb milk oligosaccharide consumptiongene cluster previously identified in B. infantis is preferentiallyinduced during growth on BMO by comparison to growth on lactose as shownin FIG. 2. These results clearly show B. infantis grown on BMO isactivated for consumption of milk oligosaccharide and a range of othergenes involved in colonization and host interface in the neonate colon,including Blon_2334, Blon_2335, Blon_2336, Blon_2337, Blon_2338,Blon_2339, Blon_2344, Blon_2346, Blon_2347, and Blon_2331, areupregulated.

Example 6. Preparation of Therapeutic Compositions for the Treatment ofPregnant Women

Preparation 1 is prepared by first diluting the ABI product of Example 4with pharmaceutical grade lactose to provide a dose of 25 billion cfu ofB. longum subsp. infantis per gram. This diluted ABI product is thenpackaged in 2-piece gel caps (1 g/gel cap) made of a gastric-resistantpolymer such as pectin, to provide doses of 25 billion cfu of activatedB. longum subsp. infantis per capsule in a delivery form that releasesits contents in the GI tract beyond the stomach.

Preparation 2 is prepared by blending the ABI product of Example 4 withthe BMO/SPF composition of Example 2 whereby 25 billion cfu of B. longumsubsp. infantis (170 mg of the dry product) is blended with 5 g of theBMO/SPF composition of Example 2. Preparation 2 provides a ratio of 25billion cfu of B. longum subsp. infantis to about 2.5 g BCO/SPF and thismixture is packaged in sachets made using a moisture and oxygenimpermeable polymer.

Example 7. Administering Composition to Pregnant Women

The composition of Preparation 1 of Example 6 (ABI) is produced and theproduct is packaged in 2-piece gel caps (1 g/gel cap) made of agastric-resistant polymer such as pectin, to provide doses of 25 billioncfu of B. longum subsp. infantis per capsule. A second preparation ismade using the dry product of Preparation 2 of Example 6 and the productis packaged in sachets made using a moisture and oxygen impermeablepolymer to provide a dose of 25 billion cfu of B. longum subsp. infantisplus 2 g BMO/SPF (5 g of the BMO/SPF composition of Example 2) persachet.

The compositions described herein are taken orally by a woman throughoutpregnancy but at least in the third trimester of pregnancy. For theinitial treatment, a pregnant woman is provided with two capsules ofPreparation 1, as described in Example 6, on a daily basis for the first2 weeks of treatment. This process establishes the population of B.longum subsp. infantis in the gut of the woman. For subsequent weeks oftreatment, the woman with is provided with 4 sachets per day ofPreparation 2 of Example 6 to maintain the population of B. longumsubsp. infantis at high levels in her gastrointestinal tract. The 4sachets are taken throughout the day, one at each meal and one beforegoing to bed. The contents of the sachet can be mixed with milk, yogurtor pudding to aid in oral consumption. If the levels of B. longum subsp.infantis fall below 25% of the levels established by the end of the2-week pre-treatment with Preparation 1 of Example 6 as determined byfecal microbiome analyses, the patient is returned to the Preparation 2treatments for an additional 2 weeks. Compositions of this inventionaccording to alternative embodiments may be administered similarly.Administration should continue until the birth of the child. Thetreatment leads to a much higher likelihood that a vaginally-deliveredinfant will be appropriately inoculated with the B. longum subsp.infantis from the mother.

Example 8. Use of a Composition Comprising Bifidobacteria with anExpressed Fucosidase to Improve the Health of an Infant

The final dry product of Example 4 (Activated Bifidobacterium longumsubsp. infantis—at a bacterial count 130×10⁹ cfu/g) is blended withpharmaceutical grade lactose to reach a bacterial count of about 25×10⁹cfu/g, and packaged into sachets made from moisture and oxygen resistantmaterials at a mass of 500 mg (12.5×10⁹ cfu) per sachet. The contents ofone sachet is provided to a newborn infant who is exclusively receivingbreast milk every day for a period of 6 months. The package is openedand the contents emptied into a small cup to which a few drops of breastmilk is added to make a watery paste. This is then provided to the babyeither using a blunt-tipped plastic dropper or the parent's fingertip.Best results are obtained if the baby consumes at least 75% of the dailycomposition each day. Monitoring of the infant fecal microbiota willindicate that the level of B. infantis in the feces will represent morethan 20% of the total microbial load of the feces. This compositionshould be provided to all babies whether they are vaginally delivered,but especially if they are delivered by Caesarian Section.

Some mothers who are nursing their babies, may be deficient in certaincomplex fucosylated oligosaccharides in their milk because of adeficiency in α1-2-fucoslyltransferase enzyme (FUC-2) as measured by agenetic test. For mothers who are of the genotype FUC-2, the sachetcontaining the ABI product of example 4 should be supplemented with aBCO composition containing synthetically produced and purifiedfucosylated oligosaccharides (SPF) at a BCO:SPF ratio of about 2:1 asdescribed in Example 2. These sachets are prepared to deliver of12.5×10⁹ cfu of ABI and 1 g BCO plus 0.5 g fucosyllactose per sachet(i.e., 100 mg of the undiluted ABI of Example 4 plus 2.5 g of BCO ofExample 2 plus 0.5 g of fucosyllactose per sachet). This composition isprovided on a daily basis for 6 months as described above for thesachets of ABI alone.

For infants that are receiving mixed feeds (breast milk and infantformula) or exclusively infant formula, the ABI product of Example 4 isblended with a bovine colostrum oligosaccharide concentrate compositionof Example 2. A composition of dried bovine colostrum oligosaccharidecontaining about 40% lactose and about 40% complex oligosaccharides(BCO) is prepared according to Example 2 and packaged in a moistureresistant sachet at a dose of 5 g/sachet (i.e., 2 g BCO/sachet)—the BMOsachet. Additional sachets containing a blend of the ABI product ofExample 4 resulting in the delivery of 12.5×10⁹ cfu of ABI, and 2 g BCOper sachet (ca. 100 mg of the undiluted ABI of Example 4 plus 5 g of BCOof Example 2)—the BLEND sachet. The contents of one BLEND sachet isprovided to a baby on a daily basis who is otherwise receiving mixedfeeds or exclusively receiving infant formula, by opening the sachet anddissolving its contents into the prepared liquid infant formula andproviding it to the baby in a morning feed. Twelve hours later theprocess is repeated but with the BMO sachet (no ABI) delivered with theinfant formula as described for the BLEND sachet. This daily cycle isrepeated (BLEND in the morning, BMO in the evening) for at least thefirst 6 months of life. Monitoring of the infant fecal microbiota willindicate that the level of B. infantis in the feces will represent morethan 20% of the total microbial load of the feces. This routine shouldbe maintained for all babies whether they are vaginally delivered, ordelivered by Caesarian Section, as long as they are not beingexclusively breast-fed. Alternatively, the infant can be provided justthe BLEND sachets twice per day providing the BLEND sachets are preparedwith only 6×10⁹ cfu of ABI per sachet.

These dietary supplements will increase the concentration of B. longumsubsp. infantis in the lower bowel of the baby to those levelshistorically seen in vaginally-delivered, breast-fed babies, and willsignificantly reduce the likelihood of a pathogenic bacterial bloom thatmay cause colic in that baby. This supplementation will alsosignificantly improve the rate of development of that baby'sgastrointestinal mucosa and mucosal immune response. Compositions ofthis invention according to alternative embodiments may be administeredsimilarly.

REFERENCES

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1-116. (canceled)
 117. A method of preparing an activatedBifidobacterium, the method comprising (a) incubating a purified strainof Bifidobacterium in a fermenter in the presence of a mammalian milkoligosaccharide, (b) harvesting activated Bifidobacterium cells, and (c)purifying the activated Bifidobacterium cells obtained in (b), whereinthe activated bifidobacterium contains a transport system capable ofinternalizing one or more oligosaccharides before said oligosaccharideis hydrolyzed and is further capable of hydrolyzing said internalizedoligosaccharide, wherein said oligosaccharide has the structure of anoligosaccharide found in a mammalian milk.
 118. The method of claim 117,wherein the activated Bifidobacterium cells express a gene coding for asialidase, a fucosidase, a sialic acid transporter, or a fucosetransporter.
 119. The method of claim 117, wherein, in the activatedBifidobacterium cells, gene Blon_0042 is upregulated, gene Blon_2168 isdownregulated, or a combination thereof.
 120. The method of claim 117,wherein any of the genes Blon_2334, Blon_2335, Blon_2336, Blon_2337,Blon_2338, Blon_2339, Blon_2344, Blon_2346, Blon_2347, and Blon_2331, isupregulated in the activated Bifidobacterium cells.
 121. The method ofclaim 117, wherein the activated Bifidobacterium cells compriseupregulated genes selected from the group consisting of Blon_0042,Blon_R0015, Blon_R0017, Blon_R0021, Blon_R0022, and combinationsthereof, and/or downregulated genes selected from the group consistingof Blon_0518, Blon_0785, Blon_2167, Blon_2168 and combinations thereof.122. The method of claim 117, wherein the Bifidobacterium is B. longum,B. breve, B. bifidum or B. pseudocatenulatum.
 123. The method of claim122, wherein the B. longum is B. longum subsp. infantis.
 124. The methodof claim 117, wherein the mammalian milk oligosaccharide is isolatedfrom a mammalian milk source.
 125. The method of claim 124, wherein themammalian milk oligosaccharide is from a human or a bovine source,optionally wherein the bovine source is bovine colostrum.
 126. Themethod of claim 117, wherein the mammalian milk oligosaccharide isselected from the group consisting of Lacto-N-Tetraose,Lacto-N-Neotetraose, Lacto-N-Fucopentaose I, Lacto-N-Fucopentaose II,Lacto-N-Fucopentaose III, Lacto-N-Fucopentaose V, Lacto-N-Hexaose,Para-Lacto-N-Hexaose, Lacto-N-Neohexaose, Para-Lacto-N-Neohexaose,Monofucosyllacto-N-Hexaose II, Isomeric Fucosylated Lacto-N-Hexaose (1),Monofucosyllacto-N-Hexaose, Isomeric Fucosylated Lacto-N-Hexaose (3),Isomeric Fucosylated Lacto-N-Hexaose (2),Difucosyl-Para-Lacto-N-Neohexaose, Difucosyl-Para-Lacto-N-Hexaose,Difucosyllacto-N-Hexaose, Lacto-N-Neoocataose, Para-Lacto-N-Octanose,Iso-Lacto-N-Octaose, Lacto-N-Octaose, Monofucosyllacto-Nneoocataose,Monofucosyllacto-N-Ocataose, Difucosyllacto-N-Octaose I,Difucosyllacto-N-Octaose 11, Difucosyllacto-N-Neoocataose 11,Difucosyllacto-N-Neoocataose 1, Lacto-N-Decaose,Trifucosyllacto-N-Neooctaose, Trifucosyllacto-N-Octaose,Trifucosyl-Iso-Lacto-N-Octaose, 2′-fucosyllactose, 3-fucosyllactose,difucosyllactose, 3′-sialyllactose, 6′-sialyllactose,3′-sialyl-3-fucosyllactose, sialyllacto-N-tetraose, and6′-sialyllactosamine.
 127. The method of claim 117, further comprisingdrying the purified activated Bifidobacterium cells.
 128. The method ofclaim 127, wherein the drying is performed by spray drying or freezedrying, optionally wherein the freeze drying is performed in thepresence of a suitable cryoprotectant, and optionally wherein thecryoprotectant is selected from the group consisting of glucose,lactose, raffinose, sucrose, trehalose, adonitol, glycerol, mannitol,methanol, polyethylene glycol, propylene glycol, ribitol, alginate,bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethylsulphoxide, sodium glutamate, glycine betaine, glycogen, hypotaurine,peptone, polyvinyl pyrrolidone, and taurine.
 129. The method of claim127, further comprising formulating the purified activatedBifidobacterium cells into a composition in the form of a dry powder,dry powder suspended in an oil, solution, packet, sachet, orallydisintegrating tablet, foodstuff, capsule, lozenge, effervescent tablet,suppository, or enema, optionally wherein the capsule or tablet has anenteric coating, and optionally wherein the enteric coating comprisesone or more of fatty acids, waxes, shellac, plastics, plant fibers,methyl acrylate-methacrylic acid copolymers, cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate, hydroxy propylmethyl cellulose acetate succinate, polyvinyl acetate phthalate (PVAP),methyl methacrylate-methacrylic acid copolymers, cellulose acetatetrimellitate, sodium alginate, and Zein.
 130. The method of claim 117,further comprising formulating the purified activated Bifidobacteriumcells into a composition such that the activated Bifidobacterium cellsare at a concentration of from 1 billion cfu/g to 500 billion cfu/g, 1billion cfu/g to 100 billion cfu/g, or 1 billion cfu/g to 50 billioncfu/g.
 131. The method of claim 127, further comprising formulating thepurified activated Bifidobacterium cells into a composition to include astabilizer, optionally wherein the stabilizer is a flow agent, andoptionally wherein the stabilizer is a milk protein.
 132. The method ofclaim 117, further comprising formulating the activated bifidobacteriumcomposition to include an isolated complex oligosaccharide.
 133. Themethod of claim 132, wherein the isolated complex oligosaccharide isisolated from a mammalian milk source selected from human, bovine, pigs,rabbits, goats, sheep, or camel, wherein the bovine source is bovinemilk, bovine colostrum, bovine colostrum concentrate, bovine wheypermeate, or mixtures thereof.
 134. The method of claim 132, wherein thecomplex oligosaccharide comprises at least one of (3Hex,4HexNac,1Fuc),(1Gal,1GlcNAc,1NeuAc), or (1Glu,1Gal,1NeuAc (3′ or 6′)), and/or whereinthe complex oligosaccharide is from bovine colostrum and comprises anyof Hex(4); Hex(4) HexNAc(2); and Hex(3) HexNAc(1) NeuAc(1) at levelsgreater than 1%, and/or wherein the complex oligosaccharide comprisesone of the following ratios of constituents: 1) a ratio of Hex(2)NeuAc(1):Hex(2) HexNAc(1) less than 5.0; 2) a ratio of Hex(2)HexNAc(1):Hex (3) HexNAc(1) of greater than 1.0; 3) a ratio of Hex(2)HexNAc(1):Hex (3) HexNAc(2) of greater than 2.0; 4) a ratio ofHex(3):Hex (3) HexNAc(1) NeuAc(1) of less than 100; or 5) a ratio ofHex(2) HexNAc(1):Hex (4) NeuAc(2) NeuGc(1) of greater than 10,optionally wherein the complex oligosaccharide is at least 20%, at least50%, or at least 80% of the weight of the composition.
 135. The methodof claim 132, wherein the complex oligosaccharide comprises afucosyllactose, sialyllactose, or derivatives thereof, optionallywherein the fucosyllactose, sialyllactose, or derivatives thereof aresynthetically produced and purified oligosaccharides of2′-fucosyllactose, 3′-fucosyllactose, difucosyllactose,lacto-N-fucosylpentose I, lacto-N-fucosylpentose II,lacto-N-fucosylpentose III, lacto-N-fucosylpentose V, 3′-sialyllactose,6′-sialyllactose, 3′-sialyl-3-fucosyllactose, sialyllacto-N-tetraose,6′-sialyllactosamine, or mixtures thereof, and/or wherein thefucosyllactose and/or sialyllactose comprises 1-5%, 5-20%, or 20-50% ofthe total oligosaccharides, and/or wherein the mass ratio of the complexoligosaccharide to the fucosyllactose and/or sialyllactose is from 20:1to 1:10, and/or wherein the complex oligosaccharide is less than 50%fucosylated.
 136. The method of claim 117, further comprisingformulating the purified activated Bifidobacterium cells into acomposition which includes a secondary metabolite, optionally whereinthe secondary metabolite is acetate, lactate, or a combinations thereof;or a short chain fatty acid.